The electrical behavior of cells and cell membranes is often of interest in basic research, as well as in modern drug development. For example, in electrophysiological experiments, electrical measurements are typically made on biological cells and cell membranes in order to understand interactions between specific membrane components. Such measurements may be performed on living cells, membranes, and/or vesicles, as well as on artificial membranes.
“Patch clamping” is one useful technique that is often used to make such measurements. FIG. 1 depicts an exemplary planar patch clamp 2 that is typically used in automated patch clamp systems. As illustrated, the planar patch clamp 2 includes a first chamber 4, a second chamber 8, and a hole 12 that fluidly connects the first and second chambers 4, 8. A biological cell 16 may be deposited inside the first chamber 4 and drawn to the hole 12, for example through the use of differential pressure applied between the first and second chambers 4, 8. A seal 20 of high electrical resistance (e.g., in the range of several hundred MΩ to greater than 1 GΩ) may then be formed between a bottom surface 24 of the first chamber 4 and the biological cell membrane 28. With such a high electrical resistance level for the seal 20 and by rendering permeable the portion 48 of the cell membrane 28 separating the first and second chambers 4, 8 (i.e., the portion 48 of the cell membrane 28 in contact with the hole 12), as described below, it is possible to isolate and measure typical physiological cell 16 currents by controlling the voltage across the cell membrane 28.
Typically, the first chamber 4 is filled with an extracellular ionic solution 32. The second chamber 8 may be filled with a saline buffer solution 34 that may contain a proper concentration of antibiotics. As illustrated, a measurement circuit is typically implemented through the use of a sensing electrode 36 in contact with the extracellular ionic solution 32, a bath electrode 40 connected to ground and in contact with the saline buffer solution 34, and an operational amplifier 44 connected to the sensing electrode 36.
Once the high-resistance seal 20 is achieved between the bottom surface 24 of the first chamber 4 and the biological cell membrane 28, the portion 48 of the cell membrane 28 separating the first and second chambers 4, 8 (i.e., the portion 48 of the cell membrane 28 in contact with the hole 12) may be permeabilized (e.g., electrically, through negative pressure, or through the use of antibiotics) so as to effectively place the bath electrode 40 inside the cell 16. This, in turn, allows for an external voltage command 52 to be placed between the sensing electrode 36 and the bath electrode 40, thereby providing for control of the cell's transmembrane voltage potential (i.e., one may achieve a voltage clamp of the cell's membrane 28). The current flowing in the measurement circuit (i.e., the current flowing through the cell membrane 28) may then be sensed by the operational amplifier 44. That current may also be subsequently recorded and analyzed by a computer controlled data acquisition and recording system 56.
Using the planar patch clamp 2 in an automated patch clamp system typically gives rise, however, to many sources of stray capacitance. Moreover, because the planar patch clamp system 2 is typically used to measure very low cell 16 current (i.e., in the pico-ampere to nano-ampere range), a very high gain resistor 58 (e.g., 10 MΩ to 10 GΩ) is often used in connection with the operational amplifier 44. As such, even very small capacitances (i.e., in the pico-farad range) that occur in the planar patch clamp 2 before the operational amplifier 44 result in very large current spikes.
Two sources of capacitance are particularly troublesome for the planar patch clamp system 2. First, it is often desirable (e.g., when scaling to larger systems having a large number of channels) to locate the operational amplifier 44 away from the first and second chambers 4, 8. Doing so, however, requires a longer cable to connect the sensing electrode 36 to the amplifier 44. But, increasing the cable length also increases the stray capacitance present in the system 2. Second, the cell membrane 28 introduces capacitive spikes into the system 2. The capacitive spikes introduced into the system 2 are much greater (e.g., up to two orders of magnitude greater), however, when multiple cells 16 are probed, rather than just a single cell 16 as illustrated in FIG. 1. Typically, the shape of the aggregate waveform of capacitive spikes that results from multiple cells 16 can not be matched and offset by a simple decay curve, thereby making it difficult to effectively compensate for such capacitive spikes using conventional methods. In addition to the cable and the cell membrane(s) 16, other sources of capacitive spikes within the planar patch clamp system 2 include, for example, electronic components (e.g., the electronics for the operational amplifier 44), printed circuit boards, connectors (e.g., relays or switches), the sensing electrode 36, and the cell holder (or chip) (i.e., the extracellular solution 32 within the first chamber 4).
Several patch clamp systems attempt to compensate for the stray capacitances introduced therein. For example, some systems employ analog compensation. In these systems, a spike having approximately the same magnitude, duration, and time constant as the capacitive spike inherent in the patch clamp system, but with inverse polarity, is generated and added to the input of the operational amplifier 44 to cancel out the inherent capacitive spike. Two benefits of this analog compensation are that the output of the amplifier 44 will not saturate from the inherent capacitive spikes, and increasing the number of amplifier channels will not increase the computational load on the computer controlled data acquisition and recording system 56 since the compensation is done in analog.
Other systems employ digital compensation. In these systems, a reference waveform is recorded, digitized, and stored. Then, during an electrophysiological experiment, the stored reference waveform is scaled and subtracted by the computer controlled data acquisition and recording system 56 following analog-to-digital conversion of the waveform output from the operational amplifier 44. While digital compensation has the benefit of requiring fewer analog components, digital compensation is typically less accurate than analog compensation over a wide range of stimulus voltages. In addition, as the number of amplifier channels increases, system performance suffers when digital compensation is employed as a greater load is placed on the computer controlled data acquisition and recording system 56. Moreover, with digital compensation, the potential for the inherent capacitive spikes to saturate the amplifier 44 output remains.
Regardless, however, of whether analog or digital compensation is employed, known patch clamp systems do not reliably and effectively verify the accuracy of the capacitance compensation, and are not flexibly designed so as to be employed in either a single-cell or multiple-cell configurations (the capacitive spikes in the system varying greatly between the two configurations).
In addition, patch clamp systems 2 that measure ionic current in biological preparations are often compromised by a series resistance (“Rs”), for example the series resistance in a measuring device such as the sensing electrode 36. Rs compensation circuits have been developed in an attempt to maintain a desired clamping voltage in the presence of this undesired series resistance.
One such exemplary Rs compensation circuit 60 is shown in FIGS. 2A and 2B. An object under test, for example the biological cell 16 having a membrane capacitance Cm, a membrane voltage Vm, a membrane current Im, and a membrane resistance Rm, is probed by the sensing electrode 36, which has a series resistance Rs, an electrode voltage Vp, and an electrode current Ip. Measurement circuitry 70, which may include the voltage clamp amplifier 44, a difference amplifier 75, and the current-to-voltage gain resistor 58, measures the electrode current Ip to produce a measured electrode current Ipmeas. A scalar 80 then multiples the measured electrode current Ipmeas to produce an Rs compensation signal Vcomp, which is then added by a voltage summer 90 to a command voltage Vc to produce a clamping voltage Vc′. As will be understood by one skilled in the art, by applying a scaled value of the measured electrode current Ipmeas as positive feedback, the effective value of the sensing electrode 36 series resistance Rs is reduced (i.e., the undesired series resistance Rs is compensated), such that the membrane voltage Vm approximately tracks the command voltage Vc even when the membrane current Im increases substantially.
A significant drawback, however, of the Rs compensation circuit 60 illustrated in FIGS. 2A and 2B is that the positive feedback loop is inherently unstable, causing undamped oscillations to occur as one begins to approach full (i.e., 100%) compensation of the undesired series resistance Rs. In reality, the undamped oscillations begin to occur even as early as once one reaches approximately 70% to 80% compensation of the undesired series resistance Rs. Accordingly, compensation of only approximately 70% to 80% of the undesired series resistance Rs using the Rs compensation circuit 60 illustrated in FIGS. 2A and 2B is achievable in practice.